SMaRT-PCR: sampling using masks and RT-PCR, a non-invasive diagnostic tool for paediatric pulmonary TB.

Key challenges in paediatric TB diagnosis are invasive sampling and poor sensitivity of standard methods. This study demonstrates the diagnostic potential of non-invasive sampling of bioaerosols from children using SMaRT-PCR, comprising mask sampling combined with reverse transcriptase-polymerase chain reaction (RT-PCR) for TB.Exhaled bioaerosols were captured on modified N-95 masks in a 10-min talk-cough-breathe process from 51 children (30 with TB confirmed using standard sampling methods and 21 without TB) aged 2-15 years. All mask samples were tested using in-house RT-PCR for 16s and rpoB RNA transcripts. Additional mask samples from children with TB were tested using Xpert® MTB/RIF (n = 3) and Xpert® MTB/RIF Ultra (n = 27).SMaRT-PCR sensitivity for detecting TB among treatment-naïve children was 96% if 16s or rpoB was present, and 75% if both genes were present, comparable to standard methods (71%) in the same cohort. Specificity was better for both genes, at 95%, than 85% for a single gene detection. Mask sampling with Xpert MTB/RIF or Ultra had a sensitivity of only 13%.This is the first study to provide evidence for testing bioaerosols as a promising alternative for detecting paediatric TB. Sampling is non-invasive and simple, with the potential for point-of-care applications. This pilot study also suggests that RNA transcript-based detection may improve TB diagnostic sensitivity in children; however, further investigation is required to establish its adaptability in clinical settings..

Identification and characterization of Aspergillus species of fruit rot fungi using microscopy, FT-IR, Raman and UV-Vis spectroscopy.

During the investigation of fungal isolation from fruit, the major genera were Aspergillus, Penicillium, cladosporium, Alternaria, fusarium, Colletotrichum were found. Among them Aspergillus (15 species) was found major dominant on different fruits. Fifteen different Aspergillus species viz. Aspergillus brasiliensis, Aspergillus phoenicis, Aspergillus carbonarius, four Aspergillus flavus, Aspergillus acidus, two Aspergillus awamori, Aspergillus aculeatus, Aspergillus eucalypticola, Aspergillus oryzae and two Aspergillus Spp. have been differentiate and identify using morphology (microscopic technique), Fourier Transforms Infrared spectroscopy (FTIR), Raman Spectroscopy (RS) and UV-visible spectrophotometry (UV-vis). The fungal mass in powder form was used in present study. In FTIR the finger print region is important for the characterization of Aspergillus because this region is unique and contains peaks indicating the presence of DNA. From the results were found Fourier transform infrared (FTIR) technique and Raman spectroscopy a useful tool, sensitive, fast, economical, accurate, not require sample preparation and successfully used to identify fungi.

Cytological behaviour of floral organs and in silico characterization of differentially expressed transcript-derived fragments associated with 'floral bud distortion' in soybean.

An attempt was made to understand the 'floral bud distortion' (FBD), an unexplored disorder prevailing in soybean. Cytological behaviour of floral reproductive organs and in silico characterization of differentially expressed transcript-derived fragments (TDFs) in symptomatic and asymptomatic soybean plants were carried out. Pollens in asymptomatic plants do not have defects in number, size, shape and function. However, in symptomatic plant, pollens were found nonviable, abnormal in shape and with reduced germination ability. Here, we employed a computational approach, exploring invaluable resources. The tissue-specific transcript profile of symptomatic and asymptomatic sources was compared to determine differentially expressed TDFs associated with FBD to improve its basic understanding. A total of 60 decamer primers produced 197 scorable amplicons, ranged 162-1130 bp, of which 171 were monomorphic and 26 were differentially regulated. Reproducible TDFs were sequenced and characterized for their homology analysis, annotation, protein-protein interaction, subcellular localization and their physical mapping. Homology-based annotation of TDFs in soybean revealed presence of two characterized and seven uncharacterized hits. Annotation of characterized sequences showed presence of genes, namely auxin response factor 9 (ARF9) and forkhead-associated (FHA) domain, which are directly involved in plant development through various pathways, such as hormonal regulation, plant morphology, embryogenesis and DNA repair.

Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7)

Purpose. We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. Methods. From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. Results. After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, 123I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. Conclusions. mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival (AU)

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Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7).

PURPOSE: We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. METHODS: From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. RESULTS: After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, (123)I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. CONCLUSIONS: mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival.

Gene-expression differences in peripheral blood between lithium responders and non-responders in the Lithium Treatment-Moderate dose Use Study (LiTMUS).

This study was designed to identify genes whose expression in peripheral blood may serve as early markers for treatment response to lithium (Li) in patients with bipolar disorder. Although changes in peripheral blood gene-expression may not relate directly to mood symptoms, differences in treatment response at the biochemical level may underlie some of the heterogeneity in clinical response to Li. Subjects were randomized to treatment with (n=28) or without (n=32) Li. Peripheral blood gene-expression was measured before and 1 month after treatment initiation, and treatment response was assessed after 6 months. In subjects treated with Li, 62 genes were differentially regulated in treatment responders and non-responders. Of these, BCL2L1 showed the greatest difference between Li responders and non-responders. These changes were specific to Li responders (n=9), and were not seen in Li non-responders or patients treated without Li, suggesting that they may have specific roles in treatment response to Li.

Prepubertal presentation of Herlyn-Werner-Wunderlich syndrome: a case report.

Herlyn-Werner-Wunderlich syndrome represents a complex female genital malformation with uterus didelphys, unilateral low vaginal obstruction, and ipsilateral renal agenesis, all 3 being secondary to mesonephric duct-induced müllerian anomalies. Clinically, this usually presents in postpubertal adolescent or adult women where hematometrocolpos produces a more pronounced mass effect and pain on the side of the obstructed hemivagina. It is extremely rare for this to present in infancy and early childhood because imaging may not show the small prepubertal uteri and vaginas clearly. We describe a 4-year-old girl with all of the features of this syndrome. Ipsilateral renal agenesis with a pelvic mass should raise the level of suspicion for this syndrome. Magnetic resonance imaging will confirm the diagnosis, so that early endoscopic resection of the obstructing vaginal septum can be carried out as definitive treatment. Long-term outcome is quite good with expectation of normal fertility.

Changes in spinach phylloepiphytic bacteria communities following minimal processing and refrigerated storage described using pyrosequencing of 16S rRNA amplicons.

AIMS: To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. METHODS AND RESULTS: Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready-to-eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10°C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to γ-Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4°C but not at 10°C storage, thus highlighting the importance of temperature in fresh packaged spinach. CONCLUSIONS: The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture- and nonculture-dependent approaches. SIGNIFICANCE AND IMPACT OF THE STUDY: Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture-based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.

Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses.

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.

Biliary atresia associated with choledochal cyst.

Choledochal cyst and biliary atresia are rare but important causes of neonatal jaundice. Both present with jaundice and acholic stool in neonatal period. Treatment and prognosis of both entities are very different. We are presenting a case in which choledochal cyst co-existed with biliary atresia; the antenatal diagnosis was that of choledochal cyst. Patient had cyst excision with hepatojejnostomy, but ultimately required portoenterostomy because of associated biliary atresia. We conclude that choledochal cyst and biliary atresia are two entities with similar antenatal and postnatal presentation; they should be differentiated as management and prognosis are different.

Urethral duplication in children: our experience of eight cases.

OBJECTIVE: Duplication of urethra has varied presentations and multiple techniques have been described for its correction. We present our experience in the management of this anomaly. MATERIAL AND METHOD: We retrospectively reviewed the records of eight patients treated for urethral duplication in 1998-2008. We managed four cases of Y-duplication, three of whom underwent buccal mucosal tube urethroplasty and one urethrourethrostomy. Two patients presented with double stream (type II A2); in one urethrourethrostomy was done and the other child underwent vesicostomy as initial treatment. Two cases were of type II B duplication (two urethra opening as single meatus); one required only dilatation of urethra and the other excision of accessory tract. RESULT: Mean age at presentation was 56.9 months. Associated anomaly was present in only two patients. Single-stage procedure was done in four patients and multi-staged procedure in three patients. Overall mean number of procedures required was 2.8; more were required in the case of Y-duplication compared to the other types. On follow-up all patients were passing urine in single stream. CONCLUSION: Thorough work up to detect any associated anomaly and type of duplication is required for the management of urethral duplication. Single-stage repair with buccal mucosa as tube urethroplasty in cases of Y-duplication is feasible with good outcome.

Rectal diaphragm in a patient with imperforate anus and rectoprostatic fistula.

The association of rectal diaphragm in an imperforate anus has not been reported until now. A 1-year-old male presented with right transverse colostomy for high anorectal malformation. The patient had imperforate anus and a recto-prostatic fistula with rectal diaphragm. We managed the case by an ano-rectal pull through with excision of the diaphragm.

The Bortnick Miller apparatus: a useful alternative to impedance audiometer for assessment of eustachian tube function in chronic suppurative otitis media.

Eustachian tube function was assessed by impedance audiometer and Bortnick Miller apparatus in 60 ears with central perforation in pars tensa. Result of both the instruments were compared and presented.

Posterior urethral valves with vesical calculus: A rare association.

Posterior urethral valve and vesical calculus are individually among the most common causes of obstructive lower urinary complaints in children. There are very few reports of association between posterior urethral valves and bladder calculus. We report three such cases. This association of the vesical calculi with posterior urethral valves may lead to a delay in the diagnosis of the posterior urethral valves. The diagnosis of posterior urethral valves should be suspected in all children with vesical or urethral calculi who have persistence of symptoms even after removal of the calculi.

CTGF, intestinal stellate cells and carcinoid fibrogenesis.

AIM: To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell. METHODS: GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay. RESULTS: Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (> 2-fold, P < 0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (> 3-fold, P < 0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P < 0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P < 0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P < 0.05) compared to non-fibrotic GI carcinoids (< 15 ng/mL). CONCLUSION: SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

A versatile computational pipeline for bacterial genome annotation improvement and comparative analysis, with Brucella as a use case.

We present a bacterial genome computational analysis pipeline, called GenVar. The pipeline, based on the program GeneWise, is designed to analyze an annotated genome and automatically identify missed gene calls and sequence variants such as genes with disrupted reading frames (split genes) and those with insertions and deletions (indels). For a given genome to be analyzed, GenVar relies on a database containing closely related genomes (such as other species or strains) as well as a few additional reference genomes. GenVar also helps identify gene disruptions probably caused by sequencing errors. We exemplify GenVar's capabilities by presenting results from the analysis of four Brucella genomes. Brucella is an important human pathogen and zoonotic agent. The analysis revealed hundreds of missed gene calls, new split genes and indels, several of which are species specific and hence provide valuable clues to the understanding of the genome basis of Brucella pathogenicity and host specificity.